Compounds which interact with the thyroid hormone receptor for the treatment of fibrotic disease

ABSTRACT

A method of alleviating a fibrotic disease selected from lung fibrosis and liver cirrhosis in a warm blooded animal, which comprises administering an effective amount of at least one compound having the formula (I)                    
     in which X stands for the oxygen or sulphur atom or for the imino (—NH—) or sulphonyl (—SO 2 —) radical, Y stands for a direct linkage, or for the oxygen or sulphur atom or for the sulphonyl (—SO 2 —) radical or for the radical of the formula —CR 1 R 2 —, wherein R 1  and R 2  which may be the same or different are hydrogen, alkyl or aryl radicals or R 1  and R 2  may be joined together to form a cycloalkyl ring, n is an integer having the value  0  or  1 , provided that when n is  0 , Y stands for the oxygen or sulphur atom or for the sulphonyl (—SO 2 —) radical, or an ester, amide or salt thereof.

This application is a continuation of U.S. application Ser. No.09/674,512 filed on Nov. 16, 2000, now U.S. Pat. No. 6,348,497 which wasthe National Stage of International Application No. PCT/GB99/01684 filedMay 27, 1999 and published in English under PCT Article 21(2).

This invention relates to fibrotic diseases.

There are many fibrotic diseases in the human. These are sometimesreferred to as chronic connective tissue diseases, and includedegradative and proliferative conditions. They include, for example,deterioration of the joints in arthritis, deformation of vessel walls inarteriosclerosis, accelerated cardiovascular problems associated withdiabetes, lung fibrosis and cirrhosis of the liver. These fibroticdiseases are generally considered and studied as separate and differentproblems. However, it is possible that common pathways exist in allthese fibrotic diseases. Indeed, corticosteriods are used in thetreatment of many of these diseases to relieve symptoms and preventassociated tissue destruction and scarring; however, the use ofcorticosteroids is associated with dose and treatment durationside-effects which limit the usefulness of this type of compound.

Some fibrotic diseases are particularly distressing, such as, forexample, those involving deterioration of cartilage and bone in thejoint. Amelioration of such diseases is a particularly difficult targetfor the pharmaceutical industry. These conditions are often accompaniedby distressing levels of pain and gradual impairment of structural bodyfunction and movement. An added difficulty in attempting to find asatisfactory cure or improvement is that joint disease appears to belargely species specific. Therefore, research into joint disease in manis made more difficult in that animal models may not be appropriate.

In mankind, joint disease is generally progressive and affects all ages,but is particularly prevalent in middle and old age. Quality of life ofthe patient is severely reduced as the level of pain and reduction inmobility increase. Treatments hitherto have, therefore, been directed atmanifestations of the disease or malfunction which are comparativelyless difficult to treat. In rheumatoid arthritis, these includesymptom-modifying anti-rheumatic drugs (so-called SMARD) for the reliefof pain and decrease in inflammation at the joint or in the synovialmembrane, such as, for example, non-steroidal anti-inflammatory drugs(so-called NSAID). Some drugs are said to be disease modifying(so-called DMARD), such as, for example, non-cytotoxics such asanti-malarials, gold, sulphasalazine, D-penicillamine, and cytotoxicssuch as cyclophosphamide and chlorambucil. However, such drug treatmentsare often associated with unpleasant or undesirable side effects, whichlimit their application and utility. A new category of anti-rheumaticdrugs, Disease Controlling Anti-Rheumatic Drugs, (so-called DCARD) hasbeen proposed to describe drugs that effectively control the destructiveprocesses but as yet no marketed anti-rheumatic drugs merit thisclassification, although corticosteroids have some inhibitory effect onthe articular damage at the low doses that can be tolerated for longterm treatment.

However, consideration of the above fibrotic diseases as a class havinga common pathway may prove valuable in attempting to invent newtreatments.

It is known that corticosteriods can regulate gene transcription bybinding to their specific receptor which is one member of the steroidreceptor super-family (Physiology of the steroid-thyroid hormone nuclearreceptor superfamily. Williams G R, Franklyn J A. Baillière's ClinicalEndocrinology and Metabolism Vol 8 No 2 pp241-266 1994). Thissuper-family includes the glucocorticoid, oestrogen, progesterone,androgen, thyroid hormone, Vitamin D and retinoic acid receptors. Suchreceptors are responsible for the regulation of many genes includingthose encoding tissue destructive proteolytic enzymes, for example,metalloproteinases (sometimes referred to as MMPs). One such MMP iscollagenase 1. Corticosteroids interact specifically with the corticoidreceptor which then binds to the promoters of the genes. This ligandedcorticosteroid receptor represses activation of the collagenase gene bymodulating the activity of transcription factor AP-1 (activating protein1, heterodimer of c-fos and c-jun proteins; reviewed by Cato and WadeBioEssays Vol 18 No 5 pp371-378 1996). The liganded steroid receptorthus modulates production of protein destructive enzymes, therebyreducing degradation of joint tissues such as cartilage and bone.

Many of the members of the superfamily of steroid receptors can modulatetissue destructive proteolytic enzyme production when liganded with thenatural hormone. However, use of the natural hormone at pharmacologicallevels is associated with severe side effects due to their biologicalactivity in many physiological systems. Corticosteroids effectivelyinhibit proteolytic enzyme production at the transcriptional level,through modulation of AP1, but the problem with corticosteroids is thatthey have additional transactivation effects on genes that lead to sideeffects, such as bone osteoporosis, diminishment of the immune responseand water retention. Some of these side effects can be life threatening.Therefore, such inhibition or modulation in the substantial absence ofthe above hitherto undesirable associated side effects would represent amajor therapeutic advance.

These undesirable additional transactivation effects are surprisinglysubstantially avoided in accordance with the present invention. It hasbeen found that the use of the compounds described below havesubstantially no transactivation properties and do not transrepress bodydefence mechanisms signalled by immune hormones such as the cytokines,IL1 and TNF and the early response transcription factor NFκB (Mukaida Net al, J Biol Chem, 269, 13289-13295, 1994). The compounds below, andtheir associated pharmaceutical compositions, are not likely, therefore,to compromise the immune system, which is one of the one of the majorproblems associated with the use of corticosteroids. This is asurprising finding and clearly separates this class of molecule from theconventional steroid hormones.

It has been found that compounds (I) hereinafter described are able toregulate MMP gene activation, but surprisingly not through occupation ofeither the corticosteroid receptor or the androgen receptor. Therefore,surprisingly compounds (I) appear to regulate MMP gene activationthrough a different receptor and hence compounds (I) can therebysubstantially prevent transcription of MMP, including collagenase 1.

Indeed, it has been further found that compounds (I) acts throughoccupation of the thyroid hormone receptor. Herein lies the basis of thepresent invention.

Thus according to the present invention, a method is provided ofalleviating fibrotic disease by regulating tissue destructiveproteolytic enzyme production in the presence of thyroid receptorbinding, but in the substantial absence of substantive corticosteroidand androgen receptor binding. This is effected by administration of aneffective amount of at least one compound having the formula (I)

in which X stands for the oxygen or sulphur atom or for the imino (—NH—)or sulphonyl (—SO₂—) radical, Y stands for a direct linkage, or for theoxygen or sulphur atom or for the sulphonyl (—SO₂—) radical or for theradical of the formula —CR¹R²—, wherein R¹ and R² which may be the sameor different are hydrogen, alkyl or aryl radicals as hereinafterdescribed, ring B may be optionally substituted by one or moresubstituents selected from halogen atoms and alkyl and aryl radicals, nis an integer having the value 0 or 1, and esters, amides and saltsthereof.

In the compounds having the formula (I), R¹ and R² may be the same ordifferent and preferably R¹ is selected from hydrogen or 1-4C alkyl, andR² from hydrogen, 1-4C alkyl or phenyl (which may be optionallysubstituted with at least one halogen atom, for example, chlorine,bromine), and R¹ and R² may be joined together to form a cycloalkyl ring(for example, cyclohexyl); the ring B may be optionally contain one ormore substituents selected from halogen atoms and 1-4C alkyl. Mostpreferably, the 1-4C alkyl radicals are methyl or ethyl, and the halogenatom is chlorine.

Esters of compound (I) may be useful in the present invention. Suchesters are preferably derived from alcohols having the formula R³—OH,where R³ is preferably 1-4C alkyl, most preferably methyl or ethyl.Salts of compound (I) include alkali metal and alkaline earth salts, andinclude magnesium, aluminium, bismuth, ammonium, and preferably sodium,potassium and calcium. Where the compound (I) contains a strongly basicsubstituent, acid addition salts thereof, such as the hydrochloride, arecomprehended.

Compounds having the formula (I), in which n is 1, and R¹ and R² whichmay be the same or different are hydrogen or alkyl radicals, aredescribed in United Kingdom patent specification 1140748, the disclosureof which is incorporated herein. Such compounds are considered to beuseful in the treatment or prophylaxis in humans and animals of suchdiseases as coronary artery disease and atherosclerosis. This is becausethey are said to reduce the concentration of cholesterol and/ortriglycerides in the blood serum and the level of fibrinogen in bloodplasma of rats. They are also said to possess anti-inflammatory activityin rats, and are, therefore, considered to be useful in the treatment ofinflammatory signs and symptoms such as rheumatoid arthritis in man;further work has shown that such compounds have substantially noanti-inflammatory properties (Billingham M E J and Rushton A,Anti-inflammatory and Anti-arthritic Drugs, Vol III, Edited by. K DRainsford, 31-63, 1985, CRC Press)).

A particularly preferred compound,1-[4-(4-chlorophenyl)benzyloxy]-1-methylpropionic acid, has the formula(II)

European patent specification 0 037 698, the disclosure of which isincorporated herein, describes processes for the production of compoundshaving the formula (I) in which n takes the value 1, Y is a direct linkand R¹ and R² which may be the same or different are hydrogen or (1-4C)alkyl. A preferred compound has the formula (III).

United Kingdom patent specification 860303, the disclosure of which isincorporated herein, describes compounds having the formula (I) in whichn takes the value 0. A preferred compound has the formula (IV) and isknown as clofibrate.

According to the present invention a method of modifying fibroticdisease in warm blooded animals is provided which comprisesadministering the animal an effective amount of at least one compoundhaving the formula (I). In a further embodiment of the present inventiona pharmaceutical composition is provided containing at least onecompound having the formula (I) for the structural modification offibrotic tissue in a warm blooded animal.

In accordance with the present invention, many fibrotic andproliferative conditions are considered to gain benefit from treatmentwith the pharmaceutical composition. These include, for example,rheumatoid arthritis, psoriatic arthritis and psoriasis itself, theloosening of prosthetic joints, atherosclerosis of cardiac and coronaryvessels and large arteries, the complications of diabetes, lungfibrosis, liver cirrhosis, systemic sclerosis, muscular dystrophy.

In order to be useful in the treatment of the above conditions inaccordance with the present invention, the above compounds may beadministered as a pharmaceutical composition by any suitable route, butpreferably orally, as, for example, tablets, capsules, suspension,emulsions, powders, syrups, elixirs. They may be administered assuppositories. The pharmaceutical compositions may be formulated toinclude any pharmaceutically acceptable excipient and may be prepared byany suitable method known on the art, such as those described in any ofthe above patent specifications. The compositions should preferably beadministered to ensure that the patient receives between 0.01 g and 0.5g of active ingredient per day; the composition containing suitablybetween 0.01 g and 0.5 g of active ingredient.

The invention is illustrated with reference to the followingexperimental information

Two plasmid constructs were introduced by standard calcium phosphateprecipitation of DNA followed by glycerol shock (Ausubel F M, Brent R,Kingston D D, Moore J G et al Current protocols in molecular biology1994 Greene Publishing Associates/Wiley-Interscience, New York, N.Y.)using a transformed African Green Monkey kidney cell line (CVl)(Schneikert J, Peterziel H, Defossez P-A, Klocker H, de Launoit Y andCato A C B Androgen receptor-Ets protein interaction is a novelmechanism for steroid hormone-mediated down-modulation of matrixmetalloproteinase expression. Journal of Biological Chemistry. Vol271(39) (pp 23907-23913), 1996).

EXPERIMENT 1

This experiment was intended to show reduction in MMP promoter activity.Accordingly, the following two plasmids were included into CV1 cellsdescribed above:

(a) Either of GR1 or pSG5ARF containing glucocorticoid or androgenreceptor (AR) respectively, both at 2 ug per 5×10⁵ cells, driven by theRous sarcoma virus promoter, and

(b) a plasmid containing the −73/+63 portion of the collagenase Ipromoter linked to luciferase gene at 8 ug per 5×10⁵ cells.

These cells were stimulated using TPA(12-O-tetradecanoylphorbol-13-acetate) at 75 ng/ml, which enhanced theactivity of the collagenase promoter. The stimulation increasedluciferase production, which was measured by luminescence after additionof the luciferase substrate (luciferin) and co-factors.

The blank represented the inclusion of these two plasmids alone withlittle endogenous activation of collagenase. Addition of TPA stimulatedthe activity of this promoter region, resulting in a high level ofluciferase production which is eventually seen as an enhancement influorescence (Y-axis). Both dihydroxytestosterone (DHT) at 10⁻⁷M anddexamethazone (Dex) at 10⁻⁷M were able to down regulate this activationthrough their respective receptors. CPII was compound II above,clobuzarit. “Empty” indicated the results when neither GR1 or ARreceptors were present. The results are presented in Table 1.

TABLE 1 Empty GR1 AR Raw % Raw % Raw % counts control counts controlcounts control Blank 2399 100 12842 100 2668 100 CPII 1882 78 6151 481374 51 TPA 5407 225 57519 448 11639 436 TPA + CPII 2747 115 39968 3115986 224 TPA + DHT 3711 139 TPA + Dex 10981 86

The above results are presented graphically in FIGS. 1a and 1 b. Fromthe results in Table 1, it is shown that Compound II was also, inaddition to DHT or Dex, able to down regulate collagenase I activation,and also to inhibit partially the background level of collagenasepromoter activity. The level of this reduction which is achieved by theuse of Compound II in both stimulated and unstimulated conditions issimilar, when expressed as ratio of luciferase counts seen withoutinclusion of this molecule, over observable counts when Compound II isincluded. This suggests that Compound II does not require the presenceof either receptor in order to exert its action. This is furthersupported by the observation that Compound II is able to reduce thelevel of both TPA stimulated and unstimulated collagenase I (MMP)promoter activity in the absence of either receptor (Empty).

EXPERIMENT 2

This experiment was intended to investigate the activation of androgenand corticosteroid receptors by compound I

The CV1 cells described above were transfected with the following twoplasmids—

(a) PGL3MMTV plasmid, containing a transcription site which is activatedby the androgen and glucocorticoid receptor, linked to the luciferasegene at 9 ug per 5×10⁵ cells, and

(b) either one of the plasmids GR1 or pSG5ARFin, containingglucocorticoid or androgen receptor (AR) respectively, under the controlof constitutively active promoters, both at 2 ug per 5×10⁵ cells.

The blank represented the inclusion of these two plasmids alone,resulting in the absence of receptor activity. Inclusion ofdihydroxytestosterone (DHT) at 10⁻⁷M or dexamethazone (Dex) at 10⁷M tothe culture medium enabled the receptor to stimulate the transcriptionof the promoter region linked to luciferase which was eventually seen asan enhancement in fluorescence (Y-axis). CPII was compound II above,clobuzarit, which was used at 10⁻⁷M.

TABLE 2 Raw counts % control AR Blank 4637 100 DHT 1731765 37347 CPII33130 714 DHT + CPU 1232241 26574 GR1 Blank 3569 100 Dex 619085 17346CPII 3272 92 Dex + CPII 748249 20965

The above results are presented graphically in FIGS. 2a and 2 b. Fromthe results in Table 2, it is shown that Compound II shows negligibleactivity to activate the same mechanism as that produced by the additionof the two steroids DHT and Dex.

EXPERIMENT 3

This experiment was intended to show the effect of compounds I on amediator of the immune response, nuclear factor −κB (NF−κB). Thefollowing two plasmids were included into CV1 cells described above—

(a) GR1 as described above at 2 ug per 5×10⁵ cells, and

(b) a plasmid designated 3-EnhTK-Luc (at 0.5 ug per 5×10⁵ cells) whichcontains a transcription site that is activated by NFκB. The cellsthemselves are capable of synthesising this protein and hence it is notnecessary to transfect this as well.

The blank represented the inclusion of these two plasmids alone; therelatively high value was caused by (i) the high level of serum in theculture, and (ii) the associated stress that the cells underwent duringthe transcription process—both of which upregulate NFκB. There was alsoa related relatively low activation of TNF (at 4 ng/ml) above a serumcontaining blank in the experiment. TNF was tumour necrosis factoralpha.

TABLE 3 Raw counts % control Blank 59538 100 CPII 75812 127 TNF 82579139 TNF + CPII 135886 228 TNF + Dex 18525 31

The above results are presented in FIG. 3. The results show thatCompound II shows little variation from the background value, indicatingthat it is not able to upregulate transcriptional activity by NFκB. Thelack of inhibitory effect of Compound II on NFκB transcription is incontrast to the marked effect of Dex, showing that Compound II does notact as an anti-inflammatory agent, so supporting the earlier clinicaldata. Also, from the lack of substantive stimulation, it can also beinferred that Compound II, in contrast to DEX, does not interfere withimmune response signalling.

EXPERIMENT 4

These experiments were intended to identify the receptor through whichcompounds I might be acting to block collagenase promoter activity.

Transfection procedure hereinbefore described with CV 1 cells wasrepeated using MCF-7 mammary breast cancer cells derived from pleuraleffusion from a breast cancer patient. Additionally, each experimentincluded transfection of a plasmid (at 8 ug per 5×10⁵ cells) containingthe −517/+63 section of the collagenase (MMP-I) promoter linked to theluciferase gene. TPA was used as above to stimulate activity of thecoliagenase promoter resulting in luciferase production. Compound II or3,3,5-triiodo-L-thyronine (T3) was included (both at 10−8M), the latterbeing a known ligand for the thyroid hormone receptor.

A series of three experiments was then carried out.

Experiment 4.1. As MCF-7 cells express endogenous thyroid hormonereceptors, only the collagenase promoter plasmid ( at 8 ug per 5×10⁵cells) was transfected into the MCF-7 cells.

The results are shown in Table 4.1.

TABLE 4.1 Raw Counts % Control Blank 12575 100 CPII 6029 47.9 TPA 1476651174.3 TPA + T3 428062 3404.1 TPA + CPII 352376 2802.2

The results in Table 4.1, presented graphically in FIG. 4.1, show thatin MCF-7 cells, both thyroid hormone and Compound II are able further toenhance TPA stimulation of luciferase production.

Experiment 4.2. The procedure of Experiment 4.1 was repeated except thata second plasmid, containing 662 bp antisense construct for the thyroidreceptor (at 8 ug per 5×10⁵ cells) was additionally transfected. Theresults are shown in Table 4.2.

TABLE able 4.2 Raw Counts % Control Blank 1458 100 CPII 2379 163.2 TPA4975 341.2 TPA + T3 19911 1365.6 TPA + CPII 8716 597.8

The results in Table 4.2, presented graphically in FIG. 4.2, show thatwhen the thyroid hormone receptor synthesis is inhibited, luciferaseproduction via the action of T3 and compound II is substantiallyprevented.

Experiment 4.3. A second plasmid was introduced containing pSP71 vector(Promega) (at 2 ug per 5×10⁵ cells) containing the coding sequence forthe chick thyroid hormone receptor which shows high homology to thehuman thyroid hormone receptor, driven by the Rous sarcoma viruspromoter to ensure constitutive production. The results are shown inTable 4.3

TABLE 4.3 Raw Counts % Control Blank 2183 100 CPII 1572 72 TPA 11528 528TPA + T3 1604 73 TPA + CPII 4414 202

The results in Table 4.3, presented graphically in FIG. 4.3, show thatCompound II, as well as thyroid hormone, is able to reduce stimulatedcollagenase transcription.

Taken together, the results of Experiments 4.1, 4.2 and 4.3 show thatcompound II mimics the action of T3 which is a known ligand for thethyroid hormone receptor.

EXPERIMENT 5

This experiment was designed to demonstrate down-regulation of pro-MMP-1and phosphorylation at serine 73 of c-jun, the latter of which is onehalf of the protein dimer AP-1, a key regulator of metalloproteaseexpression.

METHODS

Rheumatoid fibroblasts were cultured from synovium obtained from theknee joint of a patient with rheumatoid arthritis. Cells were culturedin monolayer with DMEM tissue culture medium (Gibco) with the inclusionof 5% v/v charcoal stripped serum in 75cm² flasks. The cells weretreated for 36 hours with the addition of 10 ng/ml TPA (phorbol ester)plus 5×10⁻⁷M thyroid hormone, Compound II or Compound III. TPA and drugswere added at time zero and at 30 hours, the TPA addition ensuredphosphorylation of c-jun.

After 36 hours, cells were lysed in phosphate buffered saline (PBS)containing protease inhibitors and 0.1% Triton-X-100, and then boiledfor 5 minutes in non-reducing gel loading buffer. Samples of cell lysatecorresponding to equal cell number were loaded onto a 12.5% SDSpolyacrylamide gel, and electrophoresis using a Bio-Rad™, (Bio-RadLaboratories, California USA) Mini Protean II cell was performed atapproximately 100V for one hour.

Transfer of proteins to nitrocellulose membrane was performed using aBio-Rad Mini Trans-Blot transfer cell.

Immunoblotting was performed using polyclonal rabbit anti-humanphosphorylation specific (ser 73) c-jun (cat. No 06659, UpstateBiotechnology), or MMP-1 (cat. No. RDI-MMP1Habr, Research Diagnostics)antibodies diluted 1:1000 in Tris buffered saline +3% fat free milkpowder. Swine anti-rabbit horseradish peroxidase conjugated antibody,(cat. No. p0217, Dako) also at 1:1000 dilution was used as the secondaryantibody. Chemiluminescent detection was employed using Amershamreagents according to manufacturer's instructions. Visualisation wasachieved by exposing the membrane to X-ray film (Kodak) for one hour.

Results are shown in FIG. 5. Left side column of the figure shows bandscorresponding to phosphorylated c-jun (at serine 73), complexed withDNA, at the top of the figure. Right hand side column demonstrates astrong regulated band corresponding to prod-MMP1 and a faint band at alower molecular weight corresponding to active-MMP-1 Lanes 1 and 5 cellstreated with TPA alone; Lanes 2 and 6 cells treated with TPA plusthyroid hormone; Lanes 3 and 7 cells treated with TPA plus Compound II,Lanes 4 and 8 cells treated with TPA plus Compound III.

Lanes 2, 3 and 4 show a lower intensity and therefore, amount ofphosphorylation of c-jun, when compared to the TPA alone treated controlin lane 1. Similarly, lanes 6, 7 and 8 show a lower intensity andtherefore, a lower amount of pro-MMP-1, when compared to the TPA alonetreated control in lane 5. Since the lower molecular weight band acrosslanes 5, 6, 7 and 8 corresponding to active MMP1 is very faint, theregulation by thyroid hormone and Compounds II and III appears to beoccurring at the transcriptional level to produce a similar effect tothyroid hormone on the expression of pro-MMP-1.

SUMMARY

Compound II, typical of compounds I, demonstrated steroid likeproperties in its ability to reduce collagenase promoter activity,although this was evident in the substantive absence of glucocorticoidor androgen receptors. Additionally, compound II did not activate aconstruct designed to show responsiveness to the glucocorticoidreceptor. It did not enhance or block the activity of an immune systemmediator, demonstrating that it does not possess anti-inflammatoryproperties, neither does it compromise immune response signalling. In afurther experiment, the activity of compound II was identical to T3 inthe absence and presence of endogenous or transfected thyroid hormonereceptor. This demonstrated a common mechanism for these two molecules.The liganded thyroid hormone receptor is known to be able to downregulate collagenase promoter activity (A novel mechanism of action forv-ErbA: abrogation of the inactivation of transcription factor AP-1 byretinoic acid and thyroid hormone receptors, Desbois-C; Aubert-D;Legrand-C; Pain-B; Samarut-J Cell. 1991 Nov. 15; 67(4): 731-40),although the mechanism by which this occurs has not yet been elucidated.

What is claimed is:
 1. A method of alleviating a fibrotic diseaseselected from lung fibrosis and liver cirrhosis in a warm bloodedanimal, which comprises administering an effective amount of at leastone compound having the formula (I)

in which X stands for the oxygen or sulphur atom or for the imino (—NH—)or sulphonyl (—SO₂—) radical, Y stands for a direct linkage, or for theoxygen or sulphur atom or for the sulphonyl (—SO₂—) radical or for theradical of the formula —CR¹R²—, wherein R¹ and R² which may be the sameor different are hydrogen, alkyl or aryl radicals or R¹ and R² may bejoined together to form a cycloalkyl ring, and ring B may be optionallysubstituted by one or more substituents selected from halogen atoms andalkyl and aryl radicals, when n is an integer having the value 1, or Ystands for the oxygen or sulphur atom or for the sulphonyl (—SO₂—)radical, and ring B may be optionally substituted by one or moresubstituents selected from halogen atoms and alkyl and aryl radicalswhen n has the value 0, or an ester, amide or salt thereof.
 2. A methodas claimed in claim 1 in which R¹ is selected from hydrogen and 1-4Calkyl, R²is selected from hydrogen, 1-4C alkyl and phenyl which mayoptionally be substituted by at least one halogen atom, or R¹ and R² maybe joined together to form a cyclohexyl ring, and ring B may optionallycontain one or more substituents selected from halogen atoms and 1-4Calkyl.
 3. A method as claimed in claim 1 in which the compound has theformula (II)


4. A method as claimed in claim 1 in which the compound has the formula(III)


5. A method as claimed in claim 1 in which the compound has the formula(IV)


6. A method as claimed in claim 1 in which the fibrotic disease is lungfibrosis.
 7. A method as claimed in claim 6 in which the warm bloodedanimal is a human.
 8. A method as claimed in claim 1 in which thefibrotic disease is liver cirrhosis.
 9. A method as claimed in claim 8in which the warm blooded animal is a human.